Method and products for detection of human T cell leukemia virus

ABSTRACT

A first glycoprotein having a molecular weight of approximately 61,000-68,000 daltons in the MJ, C5-MJ, C91 PL or HUT-102 cell lines, of which 46,000 to 48,000 is the unglycosylated moiety, is obtained from cells infected with human T cell leukemia virus. A second glycoprotein having a molecular weight of approximately 45,000-52,000 daltons is also obtained from such cells and is in large part identical to the NH 2  -terminal end of the first glycoprotein. The presence, in a biological specimen, of antibody to the antigenic determinant of either of these proteins is indicative of the presence of cells infected by human T cell leukemia virus. An assay for the antibody is a useful diagnostic procedure for determining such infection in biological specimens.

This invention was made in the course of work supported by the UnitedStates Government, which has certain rights in the invention.

This application is a continuation of application Ser. No. 598,673,filed Apr. 13, 1984, now U.S. Pat. No. 4,743,678, which is acontinuation-in-part of our copending application Ser. No. 489,187 filedApr. 27, 1983.

This invention relates to a novel purified forms of glycoprotein foundin the cell surface membrane of cells infected with human T cellleukemia virus, and to an assay for detecting in a biological specimenthe presence of an antibody to the antigenic determinants present insaid glycoproteins.

The human T cell leukemia virus (HTLV) is known to be closely associatedwith a particular type of human leukemia, the T cell type in adults. Ithas also been shown that all people whose bodies contain antibodies tothis virus are apparently latently infected with the virus. Essex,Journal of the National Cancer Institute, Vol. 69, 981-5 (1982). Themajor core proteins of the virus have been studied and animmunofluorescent assay procedure for antibodies to the infected cellshas been described which involves fixing cells from a cell line ofinfected human cells such as MT1 or MT2, contacting the fixed cells withthe test serum to be assayed, and determining whether bonding of theserum to the cell surface has occurred by subsequently contacting with afluorescent-labelled antibody to human IgG. Hinuma et al.,Proc.Natl.Acad.Sci., Vol. 78, 6476-6480 (1981). This assay is tediousand difficult to use because only 1 to 5% of the fixed cells display thenecessary antigenic characteristics. It has also been proposed to employa somewhat analogous cell surface immunofluorescent assay in which aculture of infected cells is incubated with test serum, then withfluorescent-labelled rabbit antibody to human IgG to determine whetherbonding of serum to cells has occurred. Robert-Guroff et al., Science,Vol. 215, 975-978 (1982). A radioimmunoassay for antibodies to p24 andp19, two of the core proteins of HTLV, has also been described. Posneret al., J.Exp.Med., Vol. 154, 333-344 (1981). However, this assay failsto produce positive results for all individuals who have been exposed tothe infecting virus and are therefore at risk, but who do not havedetectable levels of antibodies to these two antigens.

SUMMARY OF THE INVENTION

It has now been found that particular polypeptides or glycoproteinspresent on the cell surface of human T cells infected with HTLV whenpurified and isolated contain an antigenic determinant or determinantswhich provide a high degree of sensitivity and immunospecificity forantibody to human cells infected with HTLV, to human T cell leukemiacells, and/or to HTLV. Consequently, the substantially pureglycoproteins or their unglycosylated moieties are useful as adiagnostic tool for assaying biological specimens to determine whetherthey contain cells which have been infected by HTLV. Other polypeptidescontaining immunologically cross-reactive antigenic determinants areuseful for the same purpose. By "polypeptides containing immunologicallycross-reactive antigenic determinants" is meant polypeptides having incommon antigenic determinants with which a given antibody will react.Such other polypeptides include the unglycosylated moieties of theglycoproteins. Other useful polypeptides or proteins, which have thenecessary immunogenic determinants, include synthetic polypeptides. Theyalso include antibodies or fragments thereof which are anti-idiotypictowards the active determinant or determinants on the glycoprotein ofthe invention. It has recently been shown that anti-idiotypic monoclonalantibodies can induce an immune response against, and protect againstinfection by infectious organisms carrying antigenic determinants whichare the same or substantially the same as those on the anti-idiotypicantibodies (Fields el al., Nature, 300:19-23 (1982) Sacks et al.,J.Exp.Med. Vol. 155, 1108-1119 (1982)). It has also been shown thatanti-idiotypic reagents are useful as diagnostic tools for the detectionof antigens carrying sites which are immunologically cross-reactive withthose on the antibodies (Potocnjak et al., Science 215: 1637-1639(1982). Thus, an assay for HTLV could be carried out with the aid of aanti-idiotypic antibody or immunologically active fragment thereof whichcarries an antigenic site or sites thereon which are immunologicallysimilar to the antigenic site or sites on the glycoprotein of theinvention. Such anti-idiotypic antibodies can be raised against firstantibodies having specificity against the antigenic sites on theglycoprotein of the invention (i.e. the anti-idiotypic antibodies areanti-antibodies). Preferably monoclonal anti-idiotypic antibodies areused.

An assay for HTLV infection is important because the virus can bereadily transferred from the peripheral blood leukocytes ofantibody-positive people to leukocytes of antibody-negative people whenthe two are cultivated together. Popovic et al., Science, Vol. 219,856-859 (1983). Consequently, it appears that there is great risk ofinfection involved in whole blood transfusions when the transfused bloodcontains infected cells. In addition, the assay is of importance becausebiological specimens from individuals exhibiting acquiredimmunodeficient syndrome (AIDS) also give a positive test for antibodiesto the antigenic determinant of the novel glycoprotein, thusfacilitating diagnosis of that disease.

Consequently, the invention also embraces the method of assaying abiological specimen for the presence of antibody to HTLV-infected cellswhich comprises incubating said specimen with a polypeptide having anantigenic determinant or determinants immunologically cross-reactivewith those of a glycoprotein having a molecular weight of approximately61,000-68,000 daltons, of which approximately 46-48,000 daltons is theunglycosylated moiety, or with a glycoprotein having a molecular weightof approximately 45,000 to 52,000 daltons, which glycoproteins occur onthe cell surface of cells infected with HTLV, and determining whether ornot an immunocomplex is formed between said antibody and saidpolypeptide.

The invention also embraces a method of assaying a biological specimenfor the presence of antigenic determinant or determinantsimmunologically cross-reactive with the determinants of theglycoproteins of molecular weight 61,000-68,000 daltons, or45,000-52,000 daltons. The determinants to be assayed may occur on thestated glycoproteins themselves or on other polypeptides. They may be infree circulation in the body fluids or in lymphocytes. The assay can becarried out by known immunoassay methods, using antibodies, monoclonalor polyvalent, having immune reactivity with the antigenic determinantsfound on the stated glycoproteins. For example competitive immunoassaysor immunometric (sandwich) assays can be used.

The glycoproteins of the present invention have a molecular weight ofapproximately 61,000-68,000 daltons and approximately 45,000-52,000daltons as determined by sodium dodecyl sulfate (SDS) gelelectrophoresis and are soluble in SDS buffer consisting of 0.15M sodiumchloride, 0.05M Tris hydrochloride pH 7.2, 1% Triton X-100, 1% sodiumdeoxycholate, 0.1% sodium dodecylsulfate, and 1 nM phenylmethylsulfonylfluoride. Triton X-100 is a nonionic detergent (octylphenoxy polyethoxy(9-10) ethanol). The unglycosylated moiety of the 61,000-68,000 dglycoprotein has a molecular weight of approximately 46,000-48,000daltons and contains substantially the same antigenic determinant ordeterminants as does the glycoprotein itself.

The glycoproteins can be obtained from HTLV-infected cells. A variety ofcell lines have been prepared, which are permanently and persistentlyinfected with HTLV; among them can be mentioned MJ, C5-MJ, C91PL, andHUT-102. It may be that the exact sizes of the novel glycoproteins areslightly different in different lines; however, the commonimmunologically cross-reactive portion of the glycoproteins is the sameregardless of cell line, since it is a protein induced by HTLV. Thus,any cell which harbors the virus may be an appropriate source for thenovel glycoproteins In order to obtain the protein from any infectedcells carrying the virus, the cells are metabolically labelled (e.g.with ³⁵ S-methionine) and immunoprecipitated with antisera obtained fromHTLV infected subjects. The novel glycoproteins can then be detected andisolated by gel electrophoresis By "HTLV" as used in the presentspecification and claims it is meant to include the virus generically.Thus any and all forms, subtypes, or variations of the virus areincluded.

For example, the glycoproteins are present at the cell surfaces of thehuman T cell leukemia cell cultures MJ, C5-MJ, C91PL and HUT-102. Aspecimen of MJ and of C5-MJ, have been deposited with the American TypeCulture Collection (Rockville, Md.) on Apr. 26, 1983 as CRL-8294 andCRL-8293, respectively. The glycoproteins can readily be separated fromthe cells of these cell lines by lysis thereof and SDS gelelectrophoresis.

The purified and isolated glycoproteins or any antigen immunologicallycross-reactive therewith can be employed as a standard antigen in anyconventional assay procedure for detection in biological specimens ofthe presence of antibodies specific thereto, hence of the presence inthe specimen of cells infected with HTLV and/or symptomatic of AIDS. Theantibodies specific to such HTLV antigens are not found in patientssuffering from diseases such as hepatitis which are not accompanied byHTLV infection.

The glycoproteins or polypeptides immunologically cross-reactivetherewith can be labelled by conventional procedures with ¹²⁵ I or ³⁵ Sor ³ H for use in radioimmunoassay, with fluorescein for fluorescentimmunoassay, with enzyme for enzyme immunoassay or with biotin, forbiotin-avidin linked assays. It can be employed, labelled or unlabelledas desired, in competitive immunoassays, as well as in double antibodyassays using two antibodies, either of the idiotype:anti-idiotypevariety or more particularly of the second antibody type using ananti-Fc antibody, or other assays.

Alternatively, the novel glycoproteins or polypeptides immunologicallycross-reactive therewith could be immobilized on an insoluble phase,such as an insoluble resin, and detection of the anti-glycoproteinantibodies is carried out by measuring their binding to the insolublephase. Insoluble phases also include latex particles, which, when coatedwith the novel glycoprotein or its immunologically cross-reactivepolypeptides and subjected to anti-glycoprotein antibody, willagglutinate. Yet other insoluble phases include test tubes, vials,titration wells, and the like, to which the novel glycoprotein or itsimmunologically cross-reactive polypeptide can be bound, and antibodythereto detected by double antibody techniques or Protein-A dependenttechniques.

The assay for antibody to HTLV may utilize the glycoprotein orglycoproteins of m.w. 61-68,000 and 46-52,000, respectively, in crudeform, and is not limited to using these proteins in substantially pureform. For example, the glycoprotein(s) may be first substantiallypurified and then mixed together. Alternatively cruder mixtures can alsobe used. In one embodiment, the assay for the presence of antibodiesagainst HTLV may include detection of additional antibodies in thespecimen such as those against HTLV core proteins p19, p24 or a mixtureof both of the latter. In this embodiment the method comprisesincubating the specimen with a reagent comprising 1) one or bothglycoproteins of the invention (i.e. 61,000-68,000 and/or 46,000-52,000Kd or immunocross-reactive polypeptides) and, optionally, 2) HTLV coreproteins p24 or p19 or both, and determining whether or not animmunocomplex is formed between antibodies in the specimen and thereagent.

The elements necessary for carrying out the diagnostic methodologydescribed hereinbefore may be present in a kit. Such kit comprises acarrier being compartmentalized to receive therein one or morecontainers, each of said containers comprising one or more elementsnecessary to carry out the tests.

For example, the first container may contain one or both of the purifiedglycoproteins or its immunologically cross-reactive polypeptides indetectably labelled or in insolubilized form.

A second container may comprise anti IgG antibody, polyclonal ormonoclonal, useful in double antibody binding assay, or elements neededfor detection of the label on the glycoprotein or its immunologicallycross-reactive polypeptides (e.g. chromogenic substrates).

Additional containers may comprise varying amounts of one of theglycoproteins or its immunologically cross-reactive polypeptides whichcan be used to prepare a standard curve into which experimental resultscan be interpolated. The materials may be present in the kit bythemselves, in solution, freeze-dried, or in admixture with other inertmaterials, such as inert proteins, and the like.

The biological specimens tested may include blood, serum, lymphocytes,urine, tissues, saliva, feces, and the like. Of particular interest isthe screening of blood in blood banks, to assure that the blood is notcontaminated with HTLV. Screening of blood-derived products, such asvaccines, can also be done by the methods of the invention.

The following specific examples are intended to illustrate more fullythe nature of the invention without acting as a limitation upon itsscope.

EXAMPLE 1 Preparation of Labelled Glycoprotein

A. Human T cell leukemia cells from the two cell lines MJ and C5-MJ wereseparately harvested at their log phase of growth. After one wash withmethionine-free McCoy's 5A medium, each sample of the cells wasresuspended in a labelling medium consisting of methionine-free McCoy's5A, 10% phosphate-buffered saline (PBS), dialyzed fetal bovine serum,and 100 μCi/ml of ³⁵ S-methionine. At the end of a 2 to 4 hour pulsing,the radioactive labelled cells were washed three times with cold PBS.The cell pellet was then lysed with 0.6 ml to 1.0 ml of cold lysisbuffer (RIPA) (0.15M sodium chloride, 0.05M Tris-hydrochloride pH 7.2,1% Triton X-100 wetting agent, 1% sodium deoxycholate, 0.1% sodiumdodecyl sulfate, and 1 mM phenylmethylsulfonyl fluoride). After 10minutes of intermittent vortexing, the mix was centrifuged for 1 hour at100,000 xg at 4° C. The lysate supernatant was precleared of nonspecificbinding components by absorption for one hour at 4° C. on carbohydratebeads (Sepharose CL-4B) coated with protein-A.

B. Labelling of cell surface membrane protein was carried out bylactoperoxidase catalyzed radioiodination. Three aliquots of 5×10⁶ cellsof each line with greater than 99% viability were iodinated separatelywith 1 mCi of carrier-free sodium ¹²⁵ iodide in the presence of 50 μlcarrier-supported lactoperoxidase and glucosidase (Enzymobeads, Bio-RadLabs.) and 25 μl of 1% beta-D-glucose. After the reaction wasterminated, three aliquots of iodinated cells were pooled and subjectedto the same lysing and preclearing procedures as described in (A) above.

C. MJ and C5-MJ cells at their peak log phase of growth were separatelyharvested, then resuspended in glucose-free RPMI-1640 mediumsupplemented with 1 mg/ml of sodium pyruvate for 2 hours. After thisglucose starvation, the cells were labelled with 100 μCi/ml of ³H-glucosamine (New England Nuclear) for 5-6 hours. The procedure resultsin tritium labelling of only the glycoproteins present in the cells. Thelabelled cells were then subjected to lysing and preclearing proceduresas described in (A) above.

PREPARATION OF LABELLED UNGLYCOSYLATED MOIETY OF GLYCOPROTEIN

D. MJ and C5-MJ cells at their peak log phase of growth were separatelyharvested and resuspended in McCoy's 5A medium supplemented with 10%fetal bovine serum, 1% of antibiotic-antimycotic mixture, and 20 μg/mlof tunicamycin for 2 hours. After this trimming step, the cells werelabelled with 100 μCi/ml of ³⁵ S-methionine in the presence of 20 μg/mlof tunicamycin for 2 hours. The labelled material was then subjected tothe same lysing and preclearing procedures as described in (A) above.

FORMATION OF PROTEIN-ANTIBODY COMPLEX

E. There were bound to aliquots of protein-A-coated beads (a) positivereference blood serum for individual known to harbor antibodies againstcells infected with HTLV; (b) negative control serum from individualsfree from infection; and (c) serum from unknown individuals to betested. Each aliquot of coated beads was then reacted with an aliquot ofeach precleared lysate obtained from MJ and from C5-MJ from paragraph(A) above at 4° C. for 1-2 hours to permit complex formation orimmunoprecipitation to occur between the bonded lysate protein and anyantibody present in the sera. At the end of the reaction the beads werewashed 4 times with the buffer (RIPA) and once with a buffer containing0.05M Tris-hydrochloride pH 7.2 and 0.15M sodium chloride to removeuncomplexed lysate protein.

The beads were then immersed in a sample buffer (0.1M Cleland's reagent,2% sodium dodecylsulfate, 0.08M Tris-hydrochloride pH 6.8, 10% glycerol,and 0.2% Bromphenol Blue) and subjected to boiling at 100° C. for 2minutes to elute proteins from the beads and to dissociate complexes.

CHARACTERIZATION OF PROTEIN

F. Each sample from the foregoing procedure (A) was analyzed byelectrophoresis, the proteins being separated on a 12.5%SDS-polyacrylamide gel with 3.5% stacking gel using the Laemmli buffersystem. Molecular weight markers were run simultaneously in a parallelcolumn. The markers used included ¹⁴ C-labelled phosphorylase b(92,500), bovine serum albumin (68,000), and ovalbumin (46,000) carbonicanhydrase (30,000) and cytochrome C (12,000). For visualization byfluorography, the gels were first fixed with 10% acetic acid, 10%trichloroacetic acid, and 30% methanol for 1 hour, then immersed inEnhancer solution for 1 hour. After rinsing with distilled water anddrying under vacuum, the gels were exposed on Kodak SB-5 film to provideautoradiographs.

The spots of purified and isolated 61,000-68,000 molecular weightglycoprotein and of 45,000-52,000 molecular weight glycoprotein appearedprominently in all of the columns of serum (a), but in none of those ofserum (b). In contrast, the core proteins p19 and p24 failed to appearin all samples of serum (a). Appearance of the glycoprotein in theunknown sera (c) indicated the presence in the sera of cells infectedwith HTLV. Sera from individuals exhibiting AIDS also exhibitedprominent evidence of antibodies to the 61,000-68,000 MW glycoprotein inthis procedure.

Similar results have been obtained by substituting for the labelledglycoproteins of paragraph A, either of those of paragraphs B or C.There could also be substituted for it the labelled unglycosylatedmoiety of paragraph D, in which case the purified and isolated labelledmoiety appears in the gel column at a location corresponding tomolecular weight 46,000-48,000. Unlabelled glycoprotein and unlabelledunglycosylated moiety can be obtained by omitting the labelling steps inthe foregoing procedures.

Similar results have been obtained by substituting cell lines C91PL orHUT-102 in place of MJ or C5MJ.

Amino Acid Sequence Analysis.

[³⁵ S]cysteine-labeled glycoproteins having a molecular weight ofapproximately 61,000 daltons and of approximately 45,000-46,000 daltons,respectively, were precipitated from 36×10⁶ Hut 102 cells metabolicallylabeled with 5 mCi of [³⁵ S]cysteine (specific activity 1011.2 Ci/mmol,New England Nuclear) in 30 ml of cysteine-free RPMI-1640 mediacontaining 15% fetal bovine serum (Grand Island Biological Co.) for 10hours. The two glycoproteins were excised from NaDodSO₄ polyacrylamidegels and then individually subjected to electrophoretic elution in thepresence of 50 mM Tris-acetate pH 7.8 buffer containing 0.01% NaDodSO₄for 12-16 hours. Samples were dialyzed once with 10 mM of ammoniumbicarbonate buffer containing 0.002% NaDodSO₄ for 12-16 hours. One moredialysis was done with 10 mM ammonium bicarbonate buffer withoutNaDodSO₄ for 12-16 hours. Each sample was then lyophilized and aminoacid sequence analysis was conducted by the procedures described byColigan et al., J. Immun. Meth., Vol. 47, 1-11 (1981) and Meth.Enzymol., Vol. 91, 413-434 (1983). Briefly, automated Edman degradationof radiolabeled peptides was performed utilizing a Beckman 890Csequencer with cold trap modification and 0.1M Quadrol program 121078.The butyl chloride extract from each sequence step was dried using N₂evaporation in 7.0 ml scintillation vials. After addition of Biofluor(NEN), radioactivity was determined on a Beckman LS9080 liquidscintillation counter. [³⁵ S] peaks were found at residues 6, 7, 21 and28, indicating the presence of cysteine at these positions in theprotein sequence of the 61,000-68,000 d glycoprotein. The repetitiveyield for this sequence was 88%, indicating that all the residues are inthe same sequence. Small peaks at positions 4 and 13 are presumably fromsmall amounts (less than 5%) of contaminating proteins. These resultsindicate that the 61,000-68,000 mw glycoprotein is encoded, at least inpart, by the env gene of HTLV and that the leader sequence of the envgene consists of 20 residues.

Similar analysis was carried out with [³⁵ S] cysteine labeledglycoproteins having molecular weights of approximately 67,000 d and50,000-52,000 d respectively. Cysteine residues were detected atpositions 6, 7 and 21 when the first 22 NH₂ -terminal residues wereanalyzed. This shows that this glycoprotein is also encoded, at least inpart, by the same NH₂ -terminal end of the env gene.

What is claimed is:
 1. An essentially purified and essentially isolatedantigen comprising human T-cell leukemia virus type I envelopeglycoprotein having a molecular weight of approximately 61,000-68,000daltons, or determinant-containing fragments of it or of itsunglycosylated moiety, said envelope glycoprotein being characterized inthat it is present in cells infected with human T-cell leukemia virustype I and its unglycosylated moiety is approximately 46,000-48,000daltons.
 2. The antigen of claim 1 wherein said antigen is present inhuman T-cell leukemia cell line ATCC No. CRL-8294 (MJ) or ATCC No.CRL-8293 (C5-MJ).
 3. An antigen as claimed in claim 1 or 2 which isdetectably labeled.
 4. The antigen of claim 3 wherein said label isselected from the group consisting of a radiolabel, a fluorogenic label,an enzyme label, or a biotin label.
 5. The antigen of claim 1 or 2 whichis bound to an insoluble phase.
 6. The polypeptide of claim 5 whereinsaid insoluble phase is a an insoluble resin.
 7. A process for preparingthe antigen of claim 1 comprising culturing a cell-line infected withHTLV-I under appropriate conditions and extracting said antigen fromsaid cells.
 8. The method of assaying a biological specimen for thepresence of antibody indicative of invention with human T-cell leukemiavirus, type I, which method comprises:incubating said specimen with anantigen according to claim 1, said incubating occurring for a time andunder conditions sufficient to permit formation of immunocomplexesbetween said antigen and said antibody; and determining whether or notan immunocomplex is formed between said antibody and said antigen. 9.The method as claimed in claim 8 in which said antigen is saidglycoprotein having a molecular weight of approximately 61,000-68,000daltons.
 10. The method as claimed in claim 8 in which said antigen issaid unglycosylated moiety having a molecular weight of approximately46,000-48,000 daltons.
 11. The method as claimed in claim 8 whichcomprises including the step of providing said antigen in a detectablylabeled form.
 12. The method as claimed in claim 8 which is an enzymelinked immunoassay.
 13. The method as claimed in claim 8 which is aradioimmunoassay.
 14. The method as claimed in claim 8 which is a latexparticle agglutination assay.
 15. The method as claimed in claim 8 whichis a fluorescence assay.
 16. A kit useful for assaying a sample for thepresence of human antibody to cells infected with human T-cell leukemiavirus type I, said kit comprising the antigen of claim 1 in an amountsufficient for use in an immunoassay for antibody to said antigen.